Outcome and prognostic variables in childhood rhabdomyosarcoma (RMS) with emphasis on impact of FOXO1 fusions in non-metastatic RMS: experience from a tertiary cancer centre in India

While factors influencing outcomes of rhabdomyosarcoma (RMS) in developed countries have evolved from clinical characteristics to molecular profiles, similar data from developing countries are scarce. This is a single-centre analysis of outcomes in treated cases of RMS, with emphasis on prevalence, risk-migration and prognostic impact of Forkhead Box O1 (FOXO1) in non-metastatic RMS. All children with histopathologically proven RMS, treated between January 2013 and December 2018 were included. Intergroup Rhabdomyosarcoma Study-4 risk stratification was used, with treatment based on a multimodality-regimen with chemotherapy (Vincristine/Ifosfamide/Etoposide and Vincristine/Actinomycin-D/Cyclophosphamide) and appropriate local therapy. Formalin-fixed paraffin-embedded tissues were tested using Reverse Transcriptase-Polymerase Chain Reaction for FOXO1-fusions (PAX3(P3F); PAX7(P7F)). A total of 221 children (Cohort-1) were included, of which 182 patients had non-metastatic disease (Cohort-2). Thirty-six (16%), 146 (66%), 39 (18%) patients were low-risk (LR), intermediate-risk (IR) and high-risk, respectively. FOXO1-fusion status was available in 140 patients with localised RMS (Cohort 3). P3F and P7F were detected in 25/49 (51%) and 14/85 (16.5%) of alveolar and embryonal variants, respectively. The 5-year-event-free survival (EFS)/overall survival (OS) of Cohorts 1, 2 and 3 was 48.5%/55.5%, 54.6%/62.6% and 55.1%/63.7%, respectively. Amongst the localised RMS, presence of nodal metastases and primary tumour size > 10 cms were adverse prognostic factorvs (p < 0.05). On incorporating fusion-status in risk-stratification, 6/29 (21%) patients migrated from LR (A/B) to IR. All patients who re-categorised as LR (FOXO1 negative) had a 5-year EFS/OS of 80.81%/90.91%. FOXO1-negative tumours had a better 5-year relapse-free survival (58.92% versus 44.63%; p = 0.296) with a near-significant correlation in favourable-site tumours (75.10% versus 45.83%; p = 0.063). While FOXO1-fusions have superior prognostic utility compared to histology alone in localised, favourable-site RMS, traditional prognostic factors (tumour size and nodal metastases) impacted outcome the most in this subset. Strengthening of early referral systems in community and timely local intervention can help in improving outcome in resource-constrained countries.


Introduction
Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma, accounts for approximately 3.5% of childhood malignancies. With current therapy, localised disease has an over 70% 5-year event-free survival (EFS), and metastatic disease has survival rates varying from 5% to 38% [1]. Therapy in RMS is multidisciplinary, guided by site of the tumour, age of the child, extent of surgical resection, presence of distant metastases and histopathological subtype. Histopathologically, RMS is broadly divided into alveolar RMS (ARMS) and embryonal RMS (ERMS); ARMS was historically defined when any amount of biopsy component was alveolar with recent studies defining a threshold of ≥50% alveolar component to label ARMS [2]. This classification is vital as ARMS has been found to be associated with an inferior outcome when compared to its embryonal counterpart [3]. However, a biopsy may not be completely representative of the entire tumour. In addition, it is now known that ARMS lacking the characteristic Forkhead Box O1 (FOXO1) fusion has a gene expression and clinical behaviour similar to ERMS [4]. Consequently, similar to other childhood solid tumours like neuroblastoma and medulloblastoma where molecular aberrations have made their way into frontline algorithms of therapy, PAX-FOXO1 fusion status (PAX3(P3F); PAX7(P7F)) has been incorporated in contemporary risk stratification in paediatric RMS [5,6]. This retrospective study is an attempt at assessing the prevalence, risk-migration and prognostic impact of fusion status in localised childhood RMS, while also comparing the traditional prognostic variables like age, group, histological subtype, nodal status and tumour size in all treated cases of RMS at a single centre.

Eligibility criteria
The study was a retrospective audit of all children under the age of 18 years with a histopathologically confirmed diagnosis of RMS, conducted over a 6-year period from January 2013 to December 2018. Children, in whom treatment records and molecular details were available, were considered eligible for analysis. Those who had received any kind of therapy in the form of chemotherapy or radiation prior to presentation to our hospital were excluded. Children who underwent biopsy or surgical excision outside, were staged accordingly and were considered eligible. Patients with metastatic disease who received treatment were also included.

Staging and treatment
The stage, group and risk of the tumour were assigned as per the Children's Oncology Group (COG) system of risk-stratification of childhood RMS [7]. Pre-treatment evaluation of all children included a complete blood count, serum electrolytes with biochemical parameters and coagulation profile. In our study cohort, majority of our patients underwent a positron emission tomography-computed tomography (PET-CT) scan at baseline to assess both locoregional spread and distant metastases. All patients underwent bilateral bone marrow aspiration and biopsy as a part of staging. Magnetic Resonance Imaging was an adjunct imaging modality in para-meningeal, para-spinal and genitourinary tumours. In addition, children with parameningeal tumours underwent cerebrospinal fluid cytology analysis. Semen cryopreservation was offered to young adolescent boys. Orbital tumours, tumours arising in head and neck region (non-parameningeal), genitourinary tumours (non-bladder, non-prostate, non-kidney) and biliary tree were considered 'favourable site' tumours and rest were 'unfavourable site'. The tumour size was according to the largest dimension of the primary tumour reported on the pre-treatment imaging. Nodal involvement was defined as unequivocal clinico-radiological enlargement or nodal sampling [fine needle aspiration cytology (FNAC) or sampling].
The study population was analysed as three cohorts: Cohort 1 consisted of all treated cases of RMS; Cohort 2 consisted of only localised RMS; Cohort 3 consisted of all localised cases with FOXO1 fusion details. The multi-agent combination chemotherapy is outlined in Figure 1. It comprised of eight cycles of Vincristine-Ifosfamide-Etoposide (cumulative ifosfamide dose: 72 gm/m 2 ) and four cycles of Vincristine-Cyclophosphamide-Dactinomycin (cumulative cyclophosphamide dose: 8.8 gm/m 2 ). Five patients (2.3%) in the low risk (LR) (A) subset received a truncated LR protocol comprising of Vincristine, Actinomycin-D and Cyclophosphamide (cumulative cyclophosphamide dose: 4.8 gm/m 2 ) for 22 weeks [8]. After 9-12 weeks of neo-adjuvant chemotherapy, the choice and strategy for local control was finalised by a multidisciplinary planning meeting. The indications, timing and doses of radiation were administered as per institutional guidelines [9]. Following completion of treatment, children were followed up every 3 monthly in the first year, 6 monthly in the second year and yearly following that until the age of 5 years with chest radiograph at every visit and 6-monthly locoregional imaging of the primary site. Clinical examination of the primary site and evaluation of end-organ toxicity was performed at every follow-up visit.

Histology and molecular testing
Tissue for histopathology was used for histological subtyping, immunohistochemistry [Desmin, Vimentin, MyoD1, Myogenin, S100, epithelial membrane antigen (EMA), leucocyte common antigen (LCA), fli1 and Mic2] and molecular subtyping. Alveolar morphology greater than 50% was categorised as alveolar subtype. Histology was ascertained after independent reporting by two trained pathologists (SSi and MR), Formalin fixed paraffin embedded tissue containing tumour tissue was used to perform reverse transcriptase-polymerase chain reaction (RT-PCR). The technique of RNA extraction, gel electrophoresis, results, equipment, reagents and materials used in the RT-PCR, PCR mix, PCR conditions are detailed in Appendix 1.

Statistical analysis
The cohort was evaluated for both EFS and overall survival (OS). EFS was measured from the date of registration in the study until the date of the occurrence of the first event, which was designated as relapse or progression or second malignancy or death. If no event occurred, then the date of the last follow-up was used as a censored observation. OS was measured from the date of registration in the study until the date of death. In surviving patients, the date of the last follow-up was used as a censored observation. For survival analysis, all patients were censored at the date of last follow-up or date of telephonic contact. EFS and OS were computed using Kaplan-Meier method. Statistical significance of possible prognostic factors was compared using log-rank test. Multivariate analysis using Cox proportional hazards model was performed to identify risk factors and a risk model. Stata 15.0 (June 2017) was used to compute all statistical data.

Demographics
The detailed profile of the eligible patients is summarised in Table 1. A total of 397 children were diagnosed with RMS during the study period. Of these, 64 (16.1%) patients were palliated because of disseminated disease (after a multi-disciplinary team meeting and parental choice), 48 (12%) were referred outside for therapy, 44 (11%) were pre-treated and treatment details were unavailable in 20 (5%). Two hundred and twenty-one children were considered eligible for the analysis ( Figure 2) and regarded as Cohort 1. After exclusion of 39 patients (13%) with metastatic disease, 182 patients with localised disease formed Cohort 2. Of these, FOXO1 status was known in 140 patients which was designated as Cohort 3. The demographic variables and population characteristics are summarised in Table 1.

Cohort distribution
Amongst the Cohort 1, lung was the commonest site of distant metastases (isolated-17; combination-3). Bone marrow metastases were found in 9 patients (isolated-2; combination-7). Other sites of metastases were distant lymph nodes outside the regional basin, bone and liver. One patient had adrenal metastases. Cohort 2 and Cohort 3 are similar in their distribution (Table 1). One hundred and forty children comprised Cohort 3 with a boy:girl ratio of 2.1:1 and a median age of 4.4 years (1.5-16.4 years). Tumours at unfavourable sites were found in a higher frequency in girls (80%; 36/45) when compared to boys (65.3%; 62/95) (p = 0.055). Majority of our patients were in between 1 and 9 years of age (n-109; 77.9%).
The subsequent description of results is focussed on patients comprising Cohort 3 (n = 140).
As per organ of origin, the frequency of P3F & P7F was found highest in extremity tumours (14/30; 46.7%) and lowest in orbital tumours (2/11; 18%). Figure 3 is an alluvial plot showing the spectrum of site distribution of paediatric RMS as per the FOXO1 fusion status and the eventual outcome (generated online on https://rawgraphs.io/learning) [10].

Risk stratification and risk migration
As per the institutional risk stratification (adopted from the COG staging and risk stratification system) [7], the number of patients in LR (A), LR (B) and intermediate risk (IR) was 15 (10.7%), 14 (10%) and 111 (79.3%), respectively. The FOXO1 positivity rates in these groups were 26%, 14.2% and 29.7%, respectively. The current ARTS1431 risk stratification, which employs FOXO1 status in lieu of histology has shifted LR (B) and FOXO1 positive tumours under IR tumours [5]. On applying these to our cohort, 18 patients migrated to IR. Based on FOXO1 fusions alone, 6/29 (20.7%) patients migrated to IR.

Discussion
RMS is the commonest soft tissue sarcoma (STS) occurring in childhood. Factors influencing the outcome of RMS in developed countries have gradually evolved from clinical characteristics to molecular profiling but prognostic data on outcomes in RMS from developing countries are scarce. Ours is a large tertiary cancer centre and STSs form the commonest group of paediatric solid tumours, of which RMS is the most common histology (51%). A significant proportion of these patients often receive some form of treatment for symptom relief (improper surgical excision, alternative treatment) which is not cancer-directed. Because of this, they present with significant delay and with disseminated disease. In our cohort, a significant number (n = 68; 17%) with metastatic disease (to more than one site or bone/marrow metastases if single site) were palliated at presentation. This was a practical decision based on historically poor outcomes in this subset despite intensive therapy. It is also evident in this particular cohort, where amongst 39 patients with metastases who were treated (based on physician's discretion and family preference), only 11 patients (28%) were alive at last follow-up. As the decision to treat metastatic patients has been fraught with biases and is therefore non-uniform, the discussion is focussed on outcomes of all children with localised RMS who received treatment at our centre. This approach renders comparability of uniformly treated patients under our care to that, available in literature.
In an attempt to refine risk-stratification, Hibbitts et al [6], in a recently published paper, provide a consolidated review of all the prognostic variables at play in childhood RMS, across six major COG trials (D9602, D9802, D9803, ARST00331, ARST0431 and ARST0531). Our cohort had a similar gender distribution and similar proportion of infantile RMS. In contrast, our cohort had nearly twice as many patients with nodal disease (37.9% versus 18%), higher proportion of large tumours (52.9% versus 45%), more tumours at unfavourable sites (70% versus 57%) and alveolar morphology (35% versus 25%). However, on comparing our cohort with regional studies, the demographic distribution of tumours was similar [11][12][13] (Table 4).     As per the ARST1431 COG risk stratification [5] c 11 patients alive at last follow-up but not reached median time to follow-up IR RMS is in itself a heterogenous rubric of patients with outcomes ranging between 60% and 83% for different subgroups as noted on the D9803 trial [14]. Application of FOXO1 fusion status is hence an effort to fine-tune the risk stratification and hopefully guide therapy in this relatively broad subset. Preliminary data regarding impact of FOXO1 fusions was conflicting. While the Cooperative Weichteilsarkom Studiengruppe (German Sarcoma Study Group) group (Germany) did not find any difference in the outcome with regard to FOXO1 status [15], the COG studies (America) clearly demonstrated an aggressive clinical phenotype in those harbouring FOXO1 fusions [4,16]. Lack of association in the former study has been ascribed to the use of convenience cohorts (discrepancies between the outcomes of the patients whose tissue was available and not available), retrospective nature of analysis and differing treatment regimens [2]. In our cohort, the associations were strongest for traditional clinical variables like tumour size, nodal status and site of the tumour, with tumour size retaining its significance in multivariate analysis. Tumour size especially more than 10 cms had a clear adverse association with survival in our cohorts. FOXO1-positive tumours did have an inferior survival but this association was not significant on univariate analysis. In the subset of favourable tumours however, presence of FOXO1 fusions portended an inferior survival trending towards statistical significance (p = 0.063). The most recent COG publication collating data from six major trials (on 1,727 evaluable patients) has promulgated the adverse prognostic role of FOXO1 fusions as being second only to metastatic status and presently forms the cornerstone of current as well as future risk-stratification in childhood RMS [6]. Lack of such an emphatic association in our cohort can be explained by a smaller sample size. Several advantages exist on incorporating fusion studies with conventional histopathological reporting. The amount of alveolar component in a specimen to qualify it being called an ARMS continues to be a moving target [17], hence supplementing it with fusion studies allows for more objectivity. On applying the ARST1431 risk-stratification, we found that all patients (n = 11; Table 1) bracketed under the LR were surviving at the end of the study period. Aside from conventional prognostic factors and molecular profiles, there has also been interest in other factors like persistent disease metabolic activity demonstrated by PET-CT post definitive radiation being adversely associated with outcomes [18,19].
The 5-year EFS and OS in our cohort is inferior to comparable cohorts from western studies. Contributing to inferior outcomes in low-and middle-income Countries (LMIC) are several issues like large tumours at baseline, advanced stage at presentation leading to upfront palliation, inconsistent use of local control modalities like radiation and high rate of treatment abandonment [20,21]. While we have been able to curb the latter two by conducting regular multi-disciplinary meetings to ensure timely intervention and keeping in place a holistic patient tracking and support system [22], a streamlined approach of early referral of smaller/lower-stage tumours from the community is left wanting. Additionally, in LMIC setting, wherein triaging resources are important, knowledge on FOXO1 fusion helps guiding therapy and funnelling resources to the cohort that is expected to have a relatively better outcome. Being a retrospective study, the study has its limitations. A longer follow-up in these patients will shed more light on its impact on OS. Inability to offer therapy to all metastatic patients precludes identification of a subset within metastatic patients who would do better with the current approach. Therapy-wise, it was a pragmatic decision to offer a common chemotherapy backbone to all our patients, because majority of our patients belonged to IR and there was no convincing data that reducing therapy in the LR-subset would be a safe strategy in our patients. Molecular testing commenced in 2013, therefore a few patients escaped testing in the initial years. A more comprehensive testing of all paediatric RMS would elucidate the prognostic impact of FOXO1 fusion clearer. In addition, we did not test the presence of several non-FOXO1 PAX fusions like PAX-Nuclear receptor coactivator 2 (PAX-NCOA2) that may affect outcome adversely [23]. Despite these limitations, our study forms an important addition to prognostic impact of FOXO1 fusions in RMS; more so in a substantive cohort with larger tumours and higher incidence of nodal spread.

Conclusions
Fusion studies have gradually moved on from being a desirable investigation in the work-up of RMS to an essential investigation. Streamlining risk-stratification using FOXO1 fusions has teased out a smaller yet highly favourable subset within the LR (A) and LR (B) RMS, who could be candidates for de-escalation of therapy. While clinical parameters continue to be an important component of treatment algorithms, addition of molecular markers to the risk stratification can help guide therapy. In conclusion, while FOXO1 fusion does make for an exciting addition in the armamentarium to RMS risk-stratification, other modifiable factors like strengthening of early referral systems and timely local intervention can help in improving outcomes in resource-constrained countries.

Author contributions
Subramaniam Ramanathan & Sneha Sisodiya contributed equally to the work.

Data availability statement
Datasets generated during this study are available from the corresponding author on reasonable request.

Appendix 1. RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.
Total RNA was isolated from the FFPE tissue section using a Recover All Total Nucleic Acid Isolation kit (Ambion, USA). Extracted RNA was treated with RNase-free DNase I before cDNA preparation. cDNA was prepared using the H-Minus First strand cDNA synthesis system (Invitrogen). Briefly, 100 ng of total RNA was reverse transcribed into cDNA using random hexamers at 42°C for 1 hour followed by 72°C for 5 minutes. Two microlitres from the reaction was PCR amplified using PAX3 or PAX7 forward primer and FKHR reverse primer in a 20 μL reaction volume containing 10 pmol each of the forward and reverse primer, 10 μL 2× PCR master mix (Qiagen, Germany). To check the quality and integrity of the cDNA, ACTB was amplified as a housekeeping gene.
Results of molecular tests RT-PCR for PAX3-FKHR and PAX7-FKHR fusion studies were noted. Formalin-fixed paraffin-embedded tissue containing tumour tissue was used to perform RT-PCR. Equipment used in the RT-PCR, reagents and materials, PCR mix, PCR conditions is demonstrated as summarized below: Powder-free gloves Axygen 120323 All instruments were calibrated as per the standard guidelines. Positive control and negative control (Plasmid controls or known positive and negative cases) were used

Procedure
Total RNA was isolated from the FFPE tissue section using a Recover All Total Nucleic Acid Isolation kit (Ambion, USA). Extracted RNA was treated with RNase-free DNase I before cDNA preparation. cDNA was prepared using the H-Minus First strand cDNA synthesis system (Invitrogen). Briefly, 100 ng of total RNA was reverse transcribed into cDNA using random hexamers at 42°C for 1 hour followed by 72°C for 5 minutes. Two microlitres from the reaction was PCR amplified using PAX3 or PAX7 forward primer and FKHR reverse primer in a 20 μL reaction volume containing 10 pmol each of the forward and reverse primer, 10 μL 2× PCR master mix (Qiagen, Germany). To check the quality and integrity of the cDNA, ACTB was amplified as a housekeeping gene.
The master mix was aliquot in all four tubes and the contents of the tube were properly mixed and placed in the thermal cycler.

Gel electrophoresis
When the PCR was done, samples were run in 8%-10% polyacrylamide gel electrophoresis or 1.8% Agarose gel. They were stained with silver nitrate.

Sample loading in the gel
The test sample, positive, negative, and reagent control were loaded in the gel as shown in the figure below: The results were documented by taking a photograph of the gel using the gel electrophoresis unit.

Results and interpretation
The results for PAX 3/PAX7-FKHR fusion were interpreted as follows: Positive test 1) PAX3: 141 bp band position -One band appearing in between 100 and 200 bp of molecular weight marker (Lane #1). Positive control also shows the band (Lane #2) and no bands are visible in the negative sample (Lanes #3 and #4).
2) PAX7: 136 bp band position -One band appearing in between 100 and 200 bp of molecular weight marker (Lane #1). Positive control also shows the band (Lane #2). No bands were visible in the negative sample (Lanes #3 and #4).

Negative for PAX-FKHR
If there is no band in Lane #1 but positive control (Lane #2) shows band, was considered negative. PCR was repeated in case there were no bands in Lanes 3 1 and 2 or bands appeared in all the lanes.